Abstract

Pancreatic cancer is ranked the fourth most lethal cancer as less than 5% of diagnosed patients have 5 year survival or better, with median survival of only 3 to 6 months. This poor survival has been attributed, in part, to the late stage of diagnosis. Presently, several classes of drugs (anti-proliferative agents, platanating agents, fluoropyrimidines, and taxanes) are used in the treatment of this deadly disease, but patient response is poor when compared to other cancers. Therefore, there is an urgent need to improve early diagnosis as well as to identify novel molecular targets that sensitize pancreatic cancer cells to chemotherapeutic agents.

Gemcitabine (an inhibitor of DNA replication) is of particular interest to us because it remains the drug of choice in the treatment of pancreatic cancer. To our knowledge, we have recently completed the first genome-wide siRNA screen of pancreatic cancer cells to identify genes and pathways that sensitize cell killing by gemcitabine. Bioinformatics and biostatistical analysis allowed us to identify 122 strong candidate genes that we propose to validate in secondary screens. The candidates are grouped into biological pathways or networks that include cell cycle and checkpoint control, and signaling and survival pathways. In addition to the use of viability assays (CellTiterGlo) to validate our candidates, we will use high resolution time-lapse microscopy along with FACs to directly track the fates of cells. Our preliminary time-lapse studies using cell lines stably expressing gfpH2B (to visualize nuclei and chromosomes), showed that gemcitabine arrests cells in S phase for >48 hours. The prolonged checkpoint delay may allow tumor cells in patients to survive as gemcitabine has a relatively short half-life (less than 24 hours) once it is infused into the patient. Using inhibitors that abrogate the S phase checkpoint (Chk1 inhibitors) as well as Chk1 siRNA treatment following gemcitabine addition, we saw cells prematurely enter mitosis with unreplicated genomes and die. More detailed studies showed that the centromeres of all the chromosomes were physically ripped away from the bulk chromatin. Cells delay in mitosis and die, or exit mitosis with acentric chromosomes and die. This specific mitotic defect will be used as one of the assays to categorize the candidates from our siRNA screen. Our goal is to categorize candidates that kill cells through this pathway as well as candidates that kill cells through other pathways. This information will allow us to develop mechanisms by which pancreatic cancer cells can be sensitized to killing by gemcitabine. We intend to translate our discoveries towards identifying new targets for drug development as well as genetic markers to profile patient tumors to improve treatment strategies and responses.